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Synthetic antibody phage libraries

a technology of synthetic antibodies and phages, applied in the field of synthesized antibodies phage libraries, can solve the problems of inefficiency of production, reduced library size, and inability to apply in systematic and quantitative manner, and achieve high quality, high-quality target binding characteristics, and efficient generation.

Inactive Publication Date: 2005-04-14
GENENTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides methods for generating polypeptides with diversified CDRs (complementarity-determining regions) that are not dependent upon diversifying a particular CDR or a large number of amino acids. Instead, the invention proposes a systematic and objective approach to generating high-quality target-binding polypeptides with minimal structural perturbations. The invention also provides unique dimerization / multimerization techniques that further enhance the library characteristics of the polypeptides generated. The invention can be used to generate polypeptides with diverse CDRs for use in various applications, such as large and diverse pools of candidate immunoglobulin polypeptides. The invention also provides polypeptides with improved binding characteristics and faster generation times.

Problems solved by technology

However, many of these libraries have limited diversity.
The size of the library is decreased by inefficiency of production due to improper folding of the antibody or antigen binding protein and the presence of stop codons.
However, these attempts have had varying success and have not been applied in a systematic and quantitative manner.
Creating diversity in the CDR regions while minimizing the number of amino acid changes has been a challenge.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 2

Library Design: L1, L2, L3, H1, H2

[0479] Libraries of antibody variable domains were designed to maximize diversity in the CDR regions while minimizing structural perturbations in the antibody variable domains. Structural perturbations in antibody variable domains are generally associated with improperly folded antibody domains resulting in low yield, for example when produced in bacterial cells. Low yields decrease the number of binders detected in screening. Diversity in the CDR regions was generated by identifying solvent accessible and highly diverse positions in each CDR for CDRs L1, L2, L3, H1 and H2, and designing an oligonucleotide comprising at least one tailored (i.e., non-random) codon set encoding variant amino acids for the amino acid position corresponding to the position of at least one solvent accessible residue at a highly diverse position in at least one CDR region. A tailored codon set is a degenerate nucleic acid sequence that preferably encodes the most commonly...

example 3

Library Design: H3

[0490] In comparison to other CDRs, heavy chain CDR3 (H3) regions exhibit the greatest diversity in sequences and lengths, although the sequence diversity is not completely random (i.e., some amino acids were found to occur more often than other in particular amino acid positions).

[0491] In a preliminary analysis to assess the amino acid preferences for each position in H3, a library with diverse H3 was generated using an NNK codon set for residues 95-100a of the humanized antibody 4D5 H3 region. The NNK codon set encodes all 20 amino acids and stop codons. This library was generated in a Fab phage display format and 400 clones that displayed functionally on phage were identified and sequenced. The amino acid sequence found in H3 regions in the NNK library were compared to those found in the Kabat database. A comparison of those amino acids is shown in FIG. 23. When the amino acid sequences in the NNK library and Kabat database were analyzed, it was determined ther...

example 4

Construction, Sorting and Analysis of scFv Libraries

[0498] Libraries with diversified CDRs were generated using vectors comprising 4D5 variable domains in the scFv or scFv-zip formats as described in Example 1. In total, five libraries were generated and the library characteristics were as follows:

3 Library name Format CDR Diversity ScFv-1 zipper H1, H2, H3 ScFv-2 Zipper H1, H2, H3, L3 ScFv-3 Zipper H3, L3 ScFv-4 No zipper H1, H2, H3 ScFv-5 No zipper H1, H2, H3, L3

[0499] Libraries were constructed using the method of Kunkel (Kunkel et al., Methods Enzymol. (1987), 154, 367-382) with previously described methods (Sidhu et al., Methods Enzymol.(2000), 328, 333-363). For each library a "stop template" version of a scFv or scFv-zip display vector was used; in each case, a stop template with TAA stop codons within each of the CDRs to be randomized was used. Mutagenic oligonucleotides with degenerate codons at the positions to be diversified were used to simultaneously introduce CDR diver...

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Abstract

The invention provides immunoglobulin polypeptides comprising variant amino acids in CDRs of antibody variable domains. In one embodiment, the polypeptide is a variable domain of a monobody and has a variant CDRH3 region. These polypeptides provide a source of great sequence diversity that can be used as a source for identifying novel antigen binding polypeptides. The invention also provides these polypeptides as fusion polypeptides to heterologous polypeptides such as at least a portion of phage or viral coat proteins, tags and linkers. Libraries comprising a plurality of these polypeptides are also provided. In addition, methods of and compositions for generating and using these polypeptides and libraries are provided.

Description

CROSSREFERENCE TO RELATED APPLICATIONS[0001] This applications claims priority under 35 U.S.C. 119 (e) to U.S. Ser. No. 60 / 441,059 filed Jan. 16, 2003, U.S. Ser. No. 60 / 488,610, filed Jul. 18, 2003, and U.S. Ser. No. 60 / 510,314, filed Oct. 8, 2003 which are hereby incorporated by reference.[0002] The invention generally relates to libraries of antibodies or antibody variable domains. The libraries include a plurality of different antibody variable domains generated by creating diversity in the CDR regions. In particular, diversity in CDR regions is designed to maximize the diversity while minimizing the structural perturbations of the antibody variable domain. The invention also relates to fusion polypeptides of one or more antibody variable domain and a heterologous protein such as a coat protein of a virus. The invention also relates to replicable expression vectors which include a gene encoding the fusion polypeptide, host cells containing the expression vectors, a virus which di...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K16/00C12N15/13
CPCC07K16/005C07K2317/22C07K2317/54C07K2317/55C07K2319/00C07K2317/565C07K2317/567C07K2317/569C07K2317/56
Inventor BOND, CHRISTOPHER J.
Owner GENENTECH INC
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